1. Field of the Invention
This invention is in the field of medicinal chemistry. In particular, the invention relates to the use of caspase inhibitors to treat or prevent non-cancer cell death during chemotherapy and radiation therapy of cancer.
2. Description of Background Art
Organisms eliminate unwanted cells by a process variously known as regulated cell death, programmed cell death or apoptosis. Such cell death occurs as a normal aspect of animal development as well as in tissue homeostasis and aging (Glucksmann, A., Biol. Rev. Cambridge Philos. Soc. 26:59-86 (1951); Glucksmann, A., Archives de Biologie 76:419-437 (1965); Ellis et al., Dev. 112:591-603 (1991); Vaux et al., Cell 76:777-779 (1994)). Apoptosis regulates cell number, facilitates morphogenesis, removes harmful or otherwise abnormal cells and eliminates cells that have already performed their function. Additionally, apoptosis occurs in response to various physiological stresses, such as hypoxia or ischemia (PCT published application WO6/20721).
There are a number of morphological changes shared by cells experiencing regulated cell death, including plasma and nuclear membrane blebbing, cell shrinkage (condensation of nucleoplasm and cytoplasm), organelle relocalization and compaction, chromatin condensation and production of apoptotic bodies (membrane enclosed particles containing intracellular material) (Orrenius, S., J. Internal Medicine 237:529-536 (1995)).
Apoptosis is achieved through an endogenous mechanism of cellular suicide (Wyllie, A. H., in Cell Death in Biology and Pathology, Bowen and Lockshin, eds., Chapman and Hall (1981), pp. 9-34). A cell activates its internally encoded suicide program as a result of either internal or external signals. The suicide program is executed through the activation of a carefully regulated genetic program (Wylie et al., Int. Rev. Cyt. 68: 251 (1980); Ellis et al., Ann. Rev. Cell Bio. 7:663 (1991)). Apoptotic cells and bodies are usually recognized and cleared by neighboring cells or macrophages before lysis. Because of this clearance mechanism, inflammation is not induced despite the clearance of great numbers of cells (Orrenius, S., J. Internal Medicine 237:529-536 (1995)).
Mammalian interleukin-1xcex2 (IL-1xcex2) plays an important role in various pathologic processes, including chronic and acute inflammation and autoimmune diseases (Oppenheim, J. H. et. al. Immunology Today, 7, 45-56 (1986)). IL-1xcex2 is synthesized as a cell associated precursor polypeptide (pro-IL-1xcex2) that is unable to bind IL-1 receptors and is biologically inactive (Mosley et al., J. Biol. Chem. 262:2941-2944 (1987)). By inhibiting conversion of precursor IL-1xcex2 to mature IL-1xcex2, the activity of interleukin-1 can be inhibited. Interleukin-1xcex2 converting enzyme (ICE) is a protease responsible for the activation of interleukin-1xcex2 (IL-1xcex2) (Thornberry, N. A., et al., Nature 356: 768 (1992); Yuan, J., et al., Cell 75: 641 (1993)). ICE is a substrate-specific cysteine protease that cleaves the inactive prointerleukin-1 to produce the mature IL-1. The genes that encode for ICE and CPP32 are members of the mammalian ICE/Ced-3 family of genes which presently includes at least twelve members: ICE, CPP32/Yama/Apopain, mICE2, ICE4, ICHl, TX/ICH-2, MCH2, MCH3, MCH4, FLICE/MACH/MCH5, ICE-LAP6 and ICEre1III. The proteolytic activity of this family of cysteine proteases, whose active site (a cysteine residue) is essential for ICE-mediated apoptosis, appears critical in mediating cell death (Miura et al., Cell 75: 653-660 (1993)). This gene family has recently been named caspases (Alnernri, E. S. et. al. Cell, 87, 171 (1996), and Thomberry, N. A. et. al. J. Biol. Chem. 272, 17907-17911 (1997)) and divided into three groups according to its known functions. Table 1 summarizes these known caspases.
IL-1 is also a cytokine involved in mediating a wide range of biological responses including inflammation, septic shock, wound healing, hematopoiesis and growth of certain leukemias (Dinarello, C. A., Blood 77:1627-1652 (1991); diGiovine et al., Immunology Today 11:13 (1990)).
WO 93/05071 discloses peptide ICE inhibitors with the formula:
Z-Q2-Asp-Q1
wherein Z is an N-terminal protecting group; Q2 is 0 to 4 amino acids such that the sequence Q2-Asp corresponds to at least a portion of the sequence Ala-Tyr-Val-His-Asp; Q1 comprises an electronegative leaving group.
WO 96/03982 discloses aspartic acid analogs as ICE inhibitors with the formula: 
wherein R2 is H or alkyl; R3 is a leaving group such as halogen; R1 is heteroaryl-CO or an amino acid residue.
U.S. Pat. No. 5,585,357 discloses peptidic ketones as ICE inhibitors with the formula: 
wherein n is 0-2; each AA is independently L-valine or L-alanine; R1 is selected from the group consisting of N-benzyloxycarbonyl and other groups; R8, R9, R10 are each independently hydrogen, lower alkyl and other groups.
Mjalli et al. (Bioorg. Med. Chem. Lett., 3, 2689-2692, 1993) report the preparation of peptide phenylalkyl ketones as reversible inhibitors of ICE, such as: 
Thornberry et al. (Biochemistry, 33, 3934-3940, 1994) report the irreversible inactivation of ICE by peptide acyloxymethyl ketones: 
wherein Ar is COPh-2,6-(CF3)2, COPh-2,6-(CH3)2, Ph-F5 and other groups.
Dolle et al. (J. Med. Chem. 37, 563-564, 1994) report the preparation of P1 aspartate-based peptide xcex1-((2,6-dichlorobenzoyl)oxy)methyl ketones as potent time-dependent inhibitors of ICE, such as: 
Mjalli et al. (Bioorg. Med. Chem. Lett., 4, 1965-1968, 1994) report the preparation of activated ketones as potent reversible inhibitors of ICE: 
wherein X is NH(CH2)2, OCO(CH2)2, S(CH2)3 and other groups.
Dolle et al. (J. Med. Chem. 37, 3863-3866, 1994) report the preparation of xcex1-((1-phenyl-3-(trifluoromethyl)-pyrazol-5-yl)oxy)methyl ketones as irreversible inhibitor of ICE, such as: 
Mjalli et al. (Bioorg. Med. Chem. Lett., 5, 1405-1408, 1995) report inhibition of ICE by N-acyl-Aspartic acid ketones: 
wherein XR2 is NH(CH2)2Ph, OCO(CH2)2cyclohexyl and other groups.
Mjalli et al. (Bioorg. Med. Chem. Lett., 5, 1409-1414, 1995) report inhibition of ICE by N-acyl-aspartyl aryloxymethyl ketones, such as: 
Dolle et al. (J. Med. Chem. 38, 220-222, 1995) report the preparation of aspartyl xcex1-((diphenylphosphinyl)oxy)methyl ketones as irreversible inhibitors of ICE, such as: 
Graybill et al. (Bioorg. Med. Chem. Lett., 7, 41-46, 1997) report the preparation of xcex1-((tetronoyl)oxy)- and xcex1-((tetramoyl)oxy)methyl ketones as inhibitors of ICE, such as: 
Semple et al. (Bioorg. Med. Chem. Lett., 8, 959-964, 1998) report the preparation of peptidomimetic aminomethylene ketones as inhibitors of ICE, such as: 
Okamoto et al. (Chem. Pharm. Bull. 47, 11-21, 1999) report the preparation of peptide based ICE inhibitors with the P1 carboxyl group converted to an amide, such as: 
EP618223 patent application disclosed inhibitor of ICE as anti-inflammatory agents:
R-A1-A2-X-A3
Wherein R is a protecting group or optionally substituted benzyloxy; A1 is an xcex1-hydroxy or xcex1-amnino acid residue or a radical of formula: 
wherein ring A is optionally substituted by hydroxy or C1-4 alkoxy and Ra is CO or CS; A2 is an xcex1-hydroxy or xcex1-amino acid residue or A1 and A2 form together a pseudo-dipeptide or a dipeptide mimetic residue; X is a residue derived from Asp; A3 is xe2x80x94CH2xe2x80x94X1xe2x80x94COxe2x80x94Y1, xe2x80x94CH2xe2x80x94Oxe2x80x94Y2, xe2x80x94CH2xe2x80x94Sxe2x80x94Y3, wherein X1 is O or S; Y1, Y2 or Y3 is cycloaliphatic residue, and optionally substituted aryl.

WO99/18781 and U.S. Application Ser. No. 09/168,945 disclose dipeptides of formula I: 
wherein R1 is an N-terminal protecting group; AA is a residue of any natural or non-natural xcex1-amino acid, xcex2-amino acid, derivatives of an xcex1-amino acid or xcex2-amino acid; R2 is H or CH2R4 where R4 is an electronegative leaving group, and R3 is alkyl or H, provided that AA is not His, Tyr, Pro or Phe. These dipeptides are surprisingly potent caspase inhibitors of apoptosis in cell based systems. These compounds are systemically active in vivo and are potent inhibitors of antiFas-induced lethality in a mouse liver apoptosis model and have robust neuroprotective effects in a rat model of ischemic stroke. Exemplary preferred inhibitors of apoptosis include Boc-Ala-Asp-CH2F, Boc-Val-Asp-CH2F, Boc-Leu-Asp-CH2F, Ac-Val-Asp-CH2F, Ac-Ile-Asp-CH2F, Ac-Met-Asp-CH2F, Cbz-Val-Asp-CH2F, Cbz-xcex2-Ala-Asp-CH2F, Cbz-Leu-Asp-CH2F, Cbz-Ile-Asp-CH2F, Boc-Ala-Asp(OMe)-CH2F, Boc-Val-Asp(OMe)-CH2F, Boc-Leu-Asp(OMe)-CH2F, Ac-Val-Asp(OMe)-CH2F, Ac-Ile-Asp(OMe)-CH2F, Ac-Met-Asp(OMe)-CH2F, Cbz-Val-Asp(OMe)-CH2F, Cbz-xcex2-Ala-Asp(OMe)-CH2F, Cbz-Leu-Asp(OMe)-CH2F and Cbz-Ile-Asp(OMe)-CH2F.
WO 99/47154 and U.S. Application Ser. No. 09/270,735 disclose dipeptides of formula II: 
wherein R1 is an N-terminal protecting group; AA is a residue of a non-natural xcex1-amino acid or xcex2-amino acid; R2 is an optionally substituted alkyl or H. Exemplary inhibitors of caspases and apoptosis include Boc-Phg-Asp-fmk, Boc-(2-F-Phg)-Asp-fmk, Boc-(F3-Val)-Asp-fmk, Boc-(3-F-Val)-Asp-fmk, Ac-Phg-Asp-fmk, Ac-(2-F-Phg)-Asp-fmk, Ac-(F3-Val)-Asp-fmk, Ac-(3-F-Val)-Asp-fmk, Z-Phg-Asp-fmk, Z-(2-F-Phg)-Asp-fmk, Z-(F3-Val)-Asp-frnk, Z-Chg-Asp-fmk, Z-(2-Fug)-Asp-fmk, Z-(4-F-Phg)-Asp-fmk, Z-(4-Cl-Phg)-Asp-fmk, Z-(3-Thg)-Asp-fmk, Z-(2-Fua)-Asp-fmk, Z-(2-Tha)-Asp-fmk, Z-(3-Fua)-Asp-fmk, Z-(3-Tha)-Asp-fmk, Z-(3-Cl-Ala)-Asp-fmk, Z-(3-F-Ala)-Asp-fmk, Z-(F3-Ala)-Asp-fmk, Z-(3-F-3-Me-Ala)-Asp-fmk, Z-(3-Cl-3-F-Ala)-Asp-fmk, Z-(2-Me-Val)-Asp-fmk, Z-(2-Me-Ala)-Asp-fmk, Z-(2-i-Pr-xcex2-Ala)-Asp-fmk, Z-(3-Ph-xcex2-Ala)-Asp-fmk, Z-(3-CN-Ala)-Asp-fmk, Z-(1-Nal)-Asp-fmk, Z-Cha-Asp-fmk, Z-(3-CF3-Ala)-Asp-fmk, Z-(4-CF3-Phg)-Asp-fmk, Z-(3-Me2N-Ala)-Asp-fmk, Z-(2-Abu)-Asp-fmk, Z-Tle-Asp-fmk, Z-Cpg-Asp-fmk, Z-Cbg-Asp-fmk, Z-Thz-Asp-fmk, Z-(3-F-Val)-Asp-fmk, and Z-(2-Thg)-Asp-fmk; where Z is benzyloxycarbonyl, BOC is tert.-butoxycarbonyl, Ac is acetyl, Phg is phenylglycine, 2-F-Phg is (2-fluorophenyl)glycine, F3-Val is 4,4,4-trifluoro-valine, 3-F-Val is 3-fluoro-valine, 2-Thg is (2-thienyl)glycine, Chg is cyclohexylglycine, 2-Fug is (2-furyl)glycine, 4-F-Phg is (4-fluorophenyl)glycine, 4-Cl-Phg is (4-chlorophenyl)glycine, 3-Thg is (3-thienyl)glycine, 2-Fua is (2-furyl)alanine, 2-Tha is (2-thienyl)alanine, 3-Fua is (3-furyl)alanine, 3-Tha is (3-thienyl)alanine, 3-Cl-Ala is 3-chloroalanine, 3-F-Ala is 3-fluoroalanine, F3-Ala is 3,3,3-trifluoroalanine, 3-F-3-Me-Ala is 3-fluoro-3-methylalanine, 3-Cl-3-F-Ala is 3-chloro-3-fluoroalanine, 2-Me-Val is 2-methylvaline, 2-Me-Ala is 2-methylalanine, 2-i-Pr-xcex2-Ala is 3-amino-2-isopropylpropionic acid, 3-Ph-xcex2-Ala is 3-amino-3-phenylpropionic acid, 3-CN-Ala is 3-cyanoalanine, 1-Nal is 3-(1-naphthyl)-alanine, Cha is cyclohexylalanine, 3-CF3-Ala is 2-amino4,4,4-trifluorobutyric acid, 4-CF3-Phg is 4-trifluoromethylphenylglycine, 3-Me2N-Ala is 3-dimethylamino-alanine, 2-Abu is 2-aminobutyric acid, Tle is tert-leucine, Cpg is cyclopentylglycine, Cbg is cyclobutylglycine, and Thz is thioproline.
The invention arises out of the discovery that caspase inhibitors are very effective in preventing cell death induced by radiation and anticancer drugs. The invention thus relates to the use of caspase inhibitors for treating, ameliorating, and preventing non-cancer cell death during chemotherapy and radiation therapy and for treating and ameliorating the side effects of chemotherapy and radiation therapy of cancer.
In particular, the invention relates to a method of treating, ameliorating or preventing oral mucositis, gastrointestinal mucositis, bladder mucositis, proctitis, bone marrow cell death, skin cell death and hair loss resulting from chemotherapy or radiation therapy of cancer in an animal, comprising administering to the animal in need thereof an effective amount of a caspase inhibitor.